Infectious agents of prion diseases, such as Creutzfeldt Jakob Disease (CJD), are devoid of nucleic acid and instead are composed of a specific infectious protein (Prusiner (1982) Science 216:136-44). This protein, PrPSc, appears to be generated by the template-induced conformational change of a normally expressed neuronal glycoprotein, PrPC during the course of disease (Prusiner, S. B. (ed.) Prion Biology and Diseases, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999). While numerous studies have established the conversion of PrPC to PrPSc as the central pathogenic event of prion disease, cellular factors other than PrPC which may be involved in the efficient catalysis of PrPSc are unknown (Aguzzi and Weissmann Nature 389:795-8).
Various methods have been developed to enhance the amplification of PrPSc to increase the sensitivity of detecting PrPSc Saborio, et al. ((2001) Nature 411:810-3) disclose the use of a protein misfolding cyclic amplification (PMCA) method wherein prion-infected tissue homogenates containing PrPC are combined with normal brain homogenates in the presence of TRITON® X-100 and sodium dodecyl sulfate and subjected to repeated cycles of incubation and sonication to convert PrPC in normal tissue to PrPSc. Lucassen, et al. ((2003) Biochemistry 42:4127-35) disclose a modified version of the PMCA method wherein the normal and prion-infected tissue homogenates are incubated under non-denaturing conditions for the conversion of PrPC in normal tissue to PrPSc. Further, purified proteins and cell-lysate systems have been used to convert PrPC to PrPSc (Caughey, et al. (2000) Curr Issues Mol Biol 2(3):95-101; Horiuchi and Caughey (1999) Structure Fold Des. 7:R231-R240; Saborio et al. (1999) Biochem Biophys Res Commun 258:470-475). Optimal non-denaturing, cell-free conditions (KCl, MgCl2, citrate buffer and sarkosyl) for the conversion of PrPC to PrPSc have also been disclosed (Horiuchi and Caughey (1999) EMBO J. 18:3193-3203). Cordeiro, et al. ((2001) J. Biol. Chem. 276:49400-9) teach that sequence-specific DNA binding to recombinant murine prion protein converts it from PrPc to the soluble PrPSc isoform similar to that found in the fibrillar state. Further, Nandi et al. ((2002) Biochemistry 41:11017-11024) teach that the interaction between PrPc and anions (sulfate/phosphate) in polyionic ligands such as sulfated glycosaminoglycan and DNA, induce unfolding of the prion protein and conversion to PrPSc. DebBurman, et al. ((1997) Proc. Natl. Acad. Sci. USA 94(25):13938-43) demonstrate that GroEL and Hsp104 (heat shock protein 104), significantly, but distinctly affect conversion of PrPc to PrPSc.
Similarly, nucleic acids have been shown to bind to and promote the conformational change of recombinant PrP (Derrington, et al. (2002) C R Acad. Sci. III 325:17-23; Moscardini, et al. (2002) J. Mol. Biol. 318:149-59; Gabus, et al. (2001) J. Biol. Chem. 276:19301-9; Gabus, et al. (2001) J. Mol. Biol. 307:1011-21; Proske, et al. (2002) Chembiochem 3:717-25; Weiss, et al. (1997) J. Virol. 71:8790-7; Zeiler, et al. (2003) Biotechnol. Appl. Biochem. 37:173-82; Nandi, et al. (2002) J. Mol. Biol. 322:153-61; Brimacombe, et al. (1999) Biochem. J. 342:605-613).
Purified PrPC also converts into protease-resistant PrPSc in vitro in the absence of cellular cofactors (Kocisko, et al. (1995) Nature 370:471-4) and, thus, the PrP molecules themselves are sufficient to drive species- and strain-specific PrPSc formation in vitro (Bessen, et al. (1995) Nature 375:698-700; Kocisko, et al. (1995) Proc. Natl. Acad. Sci. USA 92:3923-7). However, a 50-fold molar excess of purified PrPSc is required to drive conversion of purified PrPC, suggesting that optimal efficiency of amplification may depend on the presence of cellular factors other than PrPC (Caughey, et al. (1999) Methods Enzymol. 309:122-33). Transgenic experiments in mice and cultured cells also suggest that prion formation requires a catalytic factor “X” that has high affinity for positively charged residues at the C- and N-termini of PrP (Telling, et al. (1995) Cell 83:79-90; Kanecko, et al. (1997) Proc. Natl. Acad. Sci. USA 94:10069-74; Zulianello, et al. (2000) J. Virol. 74:4351-60; Perrier, et al. (2002) Proc. Natl. Acad. Sci. USA 99:13079-84).
While PrPSc detection limits of 2 pM, corresponding to an aggregate concentration of approximately 2 fM (Bieschke, et al. (2000) Proc. Natl. Acad. Sci. USA 97(10):5468-73) to 50 pg PrPSc (Barnard, et al. (2000) Luminescence 15: 357-362) have been reported using immunoassays, improved methods of increasing the detection limits are needed to enhance the detection limits of these assays so that prion diseases may be detected at the earliest possible stages of development. It has now been found that amplification of PrPSc in vitro involves an RNA molecule that is useful for increasing the sensitivity of diagnostic methods for detecting PrPSc.